GCH1 plays a role in the high-altitude adaptation of Tibetans

2017·1.8 MB·

by  Guo, Yong-Bo

ZOOLOGICAL RESEARCH

GCH1 plays a role in the high-altitude adaptation of
Tibetans

Yong-Bo Guo1,2,#, Yao-Xi He2,4,#, Chao-Ying Cui3,#, Ouzhuluobu3, Baimakangzhuo3, Duojizhuoma3, Dejiquzong3, Bianba3,
Yi Peng2, Cai-juan Bai3, Gonggalanzi3, Yong-Yue Pan3, Qula3, Kangmin3, Cirenyangji3, Baimayangji3, Wei Guo3, Yangla3,
Hui Zhang2, Xiao-Ming Zhang2, Wang-Shan Zheng1,2, Shu-Hua Xu5,8,9, Hua Chen6, Sheng-Guo Zhao1, Yuan Cai1, Shi-Ming
Liu7, Tian-Yi Wu7, Xue-Bin Qi2,*, Bing Su2,*
1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou Gansu 730070, China
2 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming
Yunnan 650223, China
3 High Altitude Medical Research Center, School of Medicine, Tibetan University, Lhasa Tibet 850000, China
4 Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming Yunnan 650204, China
5 Chinese Academy of Sciences Key Laboratory of Computational Biology, Max Planck Independent Research Group on Population
Genomics, CAS-MPG Partner Institute for Computational Biology (PICB), Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences, Shanghai 200031, China
6 Center for Computational Genomics, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China
7 National Key Laboratory of High Altitude Medicine, High Altitude Medical Research Institute, Xining Qinghai 810012, China
8 School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China
9 Collaborative Innovation Center of Genetics and Development, Shanghai 200438, China

ABSTRACT
INTRODUCTION1
Tibetans are well adapted to high-altitude hypoxia.

Previous genome-wide scans have reported many
Tibetans are a well-known example of successful adaptation to
candidate genes for this adaptation, but only a few
extreme environments at high-altitude. Compared to lowlanders
have been studied. Here we report on a hypoxia
moving to high altitude, Tibetans show greater lung capacity,
gene (GCH1, GTP-cyclohydrolase I), involved in
function, diffusion, and ventilation, as well as lower hemoglobin
maintaining nitric oxide synthetase (NOS) function
and normal blood pressure, that harbors many (Hb) levels, better blood oxygen saturation, low hypoxic
potentially adaptive variants in Tibetans. We pulmonary vasoconstriction, high nitric oxide (NO) concentrations,
resequenced an 80.8 kb fragment covering the and lower incidence of reduced birth weight (Beall, 2006; Beall
entire gene region of GCH1 in 50 unrelated Tibetans. et al., 1997; Erzurum et al., 2007; Wu & Kayser, 2006). These
Combined with previously published data, we traits have been acquired during a long period of natural
demonstrated many GCH1 variants showing deep
divergence between highlander Tibetans and
Received: 24 March 2017; Accepted: 27 April 2017
lowlander Han Chinese. Neutrality tests confirmed a
Foundation items: This study was supported by grants from the
signal of positive Darwinian selection on GCH1 in
Tibetans. Moreover, association analysis indicated that Strategic Priority Research Program of the Chinese Academy of
the Tibetan version of GCH1 was significantly associated Sciences (XDB13010000), the National Natural Science Foundation of
with multiple physiological traits in Tibetans, including China (91631306 to BS, 31671329 to XQ, 31460287 to Ou.,
blood nitric oxide concentration, blood oxygen saturation, 31501013 to HZ and 31360032 to CC), the National 973 program
and hemoglobin concentration. Taken together, we
(2012CB518202 to TW), the State Key Laboratory of Genetic
propose that GCH1 plays a role in the genetic
Resources and Evolution (GREKF15-05, GREKF16-04), and the
adaptation of Tibetans to high altitude hypoxia.
Zhufeng Scholar Program of Tibetan University
Keywords: GCH1; Positive selection; Tibetan; #Authors contributed equally to this work
Hypoxia adaptation; Nitric oxide; Hemoglobin; *Corresponding authors, E-mail: [email protected]; [email protected]
Oxygen saturation
DOI: 10.24272/j.issn.2095-8137.2017.037
Science Press Zoological Research 38(3): 155-162, 2017 155
selection at high altitude after the ancestors of modern Tibetans
permanently settled on the Qinghai-Tibetan Plateau during the MATERIALS AND METHODS
early Upper Paleolithic period (Qi et al., 2013).
Various research groups have compared the genetic differences DNA sample collection and resequencing of GCH1 gene
between Tibetan and Han Chinese using genome-wide scans, fragment
with two key genes identified (hypoxia-inducible factor 2α, The 50 unrelated Tibetan samples were obtained from previous
HIF2α, also called EPAS1, and EGLN1) in the hypoxic pathway research (Peng et al., 2011). We resequenced an 80.8 kb
showing deep between-population divergence (Beall et al., fragment covering the gene region of GCH1 and its flanking
2010; Bigham et al., 2010; Peng et al., 2011; Simonson et al., sequences (10 kb up- and down-stream of GCH1). For
2010; Xu et al., 2011; Yi et al., 2010). For these two genes, association analysis, we collected blood samples and extracted
Tibetan-specific haplotypes have been found, which are highly DNA from 226 unrelated adult Tibetans, whose physiological data
enriched in Tibetans (~80%), but rare or absent in other world were also collected with written informed consent. The protocol of
populations (Lorenzo et al., 2014; Peng et al., 2011, 2017; this study was evaluated and approved by the Internal Review
Xiang et al., 2013). The function of selection on these two Board of Kunming Institute of China, Chinese Academy of Sciences.
genes have been shown to cause blunted physiological
responses under high altitude hypoxic conditions (Lorenzo et al., Detection of selection on GCH1 in Tibetans
2014; Peng et al., 2017; Xiang et al., 2013). For example, the The initially identified sequence variants were filtered by
Tibetan versions of EPAS1 and EGLN1 protect Tibetans from removing single nucleotide polymorphisms (SNPs) showing a
the over production of red blood cells (polycythemia), a significant deviation from the Hardy-Weinberg Equilibrium
common deleterious physiological response when lowlanders (HWE<0.000 1), and those with an excessive missing genotype
migrate to high altitude areas (Beall et al., 2010; Erzurum et al., rate (MGR>0.05). Four methods were used for selection testing,
2007; Lorenzo et al., 2014; Peng et al., 2011, 2017; Petousi et including two allele-frequency-based and two haplotype-based
al., 2014; Xiang et al., 2013; Xu et al., 2011; Zhang et al., 2010). tests. Locus specific F was calculated between 83 Tibetans
ST
Many genes are likely involved in complex traits like high and three reference populations (103 Han Chinese, CHB; 99
altitude adaptation. In addition to EPAS1 and EGLN1, previous Europeans, CEU; and 108 Africans, YRI) following Weir &
genome-wide studies have identified other candidate genes Cockerham (1984). The Tajima’s D-test was also performed
that might also contribute to genetic adaptation in Tibetans following the published procedure (Tajima, 1989).
(Beall et al., 2010; Bigham et al., 2010; Peng et al., 2011; The two haplotype-based tests included the iHS and XP-EHH
Simonson et al., 2010; Xu et al., 2011; Yi et al., 2010). The tests (Sabeti et al., 2002). The iHS score was calculated for
GTP-cyclohydrolase I (GCH1) gene is a reported candidate that each site using selscan (Szpiech & Hernandez, 2014) based on
shows relatively deep allelic divergence between Tibetans and the phased haplotypes, and only those allelic loci whose
Han Chinese, a sign of positive Darwinian selection (Peng et al., ancestral alleles were known with certainty were included in the
2011). GCH1 is located on human chromosome 14 (14q22.1- analysis (Voight et al., 2006). The XP-EHH (Sabeti et al., 2007)
q22.2), spanning 60.8 kb with six exons. Furthermore, GCH1 is analysis was used to detect the extended haplotypes due to
a rate-limiting enzyme in the de novo synthesis of positive selection. Han Chinese were used as the reference
tetrahydrobiopterin (BH4). It has been reported that under population in the XP-EHH test. We computed XP-EHH scores
hypoxia, GCH1 can promote cancer growth, and its using selscan (Szpiech & Hernandez, 2014) based on phased
expression and that of endothelial nitric oxide synthetase haplotypes of Tibetans and Han Chinese. The XP-EHH score of
(eNOS) is upregulated (Pickert et al., 2013). GCH1 is each SNP was standardized by the mean XP-EHH and the
considered as a major factor in maintaining nitric oxide standard deviation (SD) over the genome.
synthetase (NOS) function and normal blood pressure, and its
inhibition can increase blood pressure due to NOS uncoupling, Functional prediction of GCH1 candidate SNPs
which is found in many cardiovascular diseases such as Functional enrichment analyses of the candidate variants were
hypertension and atherosclerosis (Antoniades et al., 2006). performed using the Combined Annotation Dependent
Hence, the known functional role of GCH1 also makes it a Depletion (CADD) database (http://krishna.gs.washington.edu/
candidate for high-altitude adaptation in Tibetans. download/CADD/v1.3/1000G_phase3_inclAnno.tsv.gz), which
We resequenced an 80.8 kb fragment covering the entire incorporates data from ENCODE (http://genome.ucsc.edu/
gene region of GCH1 in 50 unrelated Tibetans. Combined with ENCODE/) and NIH Roadmap Epigenomics using ChromHMM
published data, we found signals of positive selection on GCH1 (https://sites.google.com/site/anshulkundaje/projects/epigenom
in Tibetans, with multiple sequence variants showing deep e roadmap#TOC-Core-Integrative-chromatin-state-maps-127-
genetic divergence between highlander Tibetans and lowlander Epigenomes-) (Ernst & Kellis, 2012).
Han Chinese. Genetic association analysis detected significant Evolutionary constraint is an indication of functional
correlation of the GCH1 variants with multiple physiological importance. We used Genome Evolutionary Rate Profiling
traits of Tibetans, including blood nitric oxide (NO) concentration, (GERP) to evaluate how conserved a test SNP was compared
blood oxygen saturation level, and hemoglobin concentration. with SNP-containing sequences from different species (http://
Hence, GCH1 might play an essential role in high-altitude mendel.stanford.edu/SidowLab/downloads/gerp/). The GERP++
adaptation in Tibetans. method was used to calculate site-specific RS scores (Davydov
156 www.zoores.ac.cn
et al., 2010). A positive GERP++ score indicates evolutionary For physiological parameters, we collected hemoglobin (Hb)
constraint, and the greater the score, the greater the level of concentration, arterial oxygen saturation (SaO) level, and
2
evolutionary constraint inferred to be acting on that site. blood nitric oxide concentration, representing key adaptive
The H3K4Me1 value indicates the maximum ENCODE H3K4 physiological traits in Tibetans (Wu & Kayser, 2006).
methylation level (maximum value observed across 16 The Hb level in blood was measured using a HemoCue Hb
ENCODE cell lines at a given position), suggestive of an 201+ analyzer (Angelholm, Sweden). The SaO level was
2
enhancer or other regulatory activities. The H3K4Me3 value measured using fingertip blood with a hand-held pulse oximeter
indicates the maximum ENCODE H3K4 trimethylation level, (Nellcor NPB-40, CA, USA). Blood NO was measured using a
and is an indication of a promoter. The DNase P value indicates nitric oxide analyzer (Sievers Model-280, GE Analytical
evidence for open chromatin. The transcription factor binding Instruments; Boulder, CO, USA).
site (TFBS) is indicated by the number of different overlapping
ChIP transcription factor binding sites. In addition, the splice site SNP genotyping and association analysis
results indicate if the tested variant is within an ACCEPTOR or We selected nine tag SNPs, covering the entire gene region of
a DONOR (Supplementary Table S1). GCH1 (Table 1). Genotyping was conducted by SNaPshot on
an ABI 3130 sequencer (Applied Bio-Systems, Forster City, CA,
Measurements of physiological traits USA). Genetic association analysis was executed using PLINK
Physiological data and blood samples were collected from 226 1.07 (Purcell et al., 2007). We used an additive model in the
unrelated Tibetans permanently residing in Bange county association analysis as all candidate SNPs were non-coding
(n=135, 37.41±3.8 years old) at an elevation of 4 700 m and and likely influenced the level of gene expression. Permutations
Lhasa city (n=91, 35.33±6.8 years old) at an elevation of 3 600 (100 000 times for each test) were performed for statistical
m. Written informed consent from all participants was obtained. assessment and correction for multiple tests.
Table 1 Association of nine GCH1 variants with three physiological traits in Tibetans
Male (n=91) Female (n=135) All (n=226)
Traits SNP_ID
Beta EMP' Beta EMP' Beta EMP'' R2 (%)
Hb rs7148266 –4.33 0.48 –0.53 0.78 –1.51 0.78 0.05
rs17128004 –3.02 0.65 –0.49 0.86 –1.10 0.86 0.01
rs4411417 –4.33 0.48 –1.27 0.86 –1.85 0.69 0.23
rs2183082 1.17 0.86 –2.10 0.57 –0.65 0.86 0.08
rs10220344 1.17 0.86 –2.10 0.57 –0.65 0.86 0.08
rs146540091 –62.95 0.05 –17.31 0.25 –35.15 0.02 1.65
rs117863726 –62.95 0.05 –17.61 0.47 –36.27 0.02 2.39
rs10136972 12.97 0.03 0.21 1.00 4.28 0.28 1.29
rs112700866 22.04 0.05 –0.78 1.00 10.25 0.16 0.67
NO rs7148266 –15.97 0.04 –10.82 0.04 –12.96 2.32E-03 3.05
rs17128004 –17.23 0.03 –10.77 0.04 –13.42 2.32E-03 3.08
rs4411417 –15.97 0.04 –11.67 0.04 –13.63 2.75E-03 3.76
rs2183082 –6.35 0.54 –6.05 0.17 –6.32 0.08 1.28
rs10220344 –6.35 0.54 –6.05 0.17 –6.32 0.08 1.28
rs146540091 –162.70 3.53E–03 13.14 0.78 –41.65 0.07 3.36
rs117863726 –162.70 3.53E–03 13.07 0.65 –41.05 0.07 1.40
rs10136972 –5.71 0.65 –14.60 0.01 –11.35 0.02 1.88
rs112700866 –10.98 0.35 4.97 0.73 –2.78 0.86 0.03
SaO2 rs7148266 –0.84 0.38 0.55 0.78 0.05 0.86 0.01
rs17128004 –1.08 0.33 –0.05 1.00 –0.44 0.48 0.07
rs4411417 –0.84 0.38 0.57 0.65 0.03 1.00 2.50E-03
rs2183082 –0.50 0.57 –0.38 0.78 –0.44 0.52 0.03
rs10220344 –0.50 0.57 –0.38 0.78 –0.44 0.52 0.03
rs146540091 12.53 0.05 –1.61 0.78 3.07 0.54 3.22
rs117863726 12.53 0.05 6.66 0.14 8.53 0.02 2.47
rs10136972 –1.86 0.27 –1.84 0.11 –1.84 0.05 0.75
rs112700866 –1.68 0.67 –3.46 0.17 –2.76 0.16 0.75
Hb, hemoglobin concentration; NO, blood nitric oxide concentration; SaO, blood oxygen saturation level. EMP’, P value after multiple test corrections;
2
EMP’’, P value after multiple test corrections with sex as the covariant.
Zoological Research 38(3): 155-162, 2017 157
adaptive alleles having lower Hb levels, consistent with
RESULTS previous results for EPAS1 and EGLN1 (Peng et al., 2011, 2017;
Xiang et al., 2013). Another SNP (rs137863726) was
Resequencing of GCH1 in Tibetans and tests of selection associated with blood oxygen saturation, with the adaptive
Previous DNA array-based genome-wide studies have only allele exhibiting higher SaO levels.
2
covered a limited number of GCH1 sequence variants (Peng et For the four SNPs (rs7148266, rs17128004, rs4411417, and
al., 2011). To obtain complete sequence data of GCH1, we first rs10136972) showing significant association with blood NO
resequenced an 80.8 kb fragment covering the entire gene levels, functional prediction analysis indicated that they were
region of GCH1 (60.8 kb) as well as the flanking sequences (10 located in the GCH1 intron regions with peak signals for
kb from upstream and 10 kb from downstream regions). In total, H3K4Me1, H3K4Me3, DNase-I, and TFBS. For example, the
we sequenced 50 unrelated Tibetan individuals, as reported H3K4Me1 peak values for rs10136972 and rs4411417 were 8.2
previously (Peng et al., 2011). In addition, we obtained the and 5.9, respectively, indicating their potential role in gene
GCH1 sequences of 33 Tibetans from recently published whole expression regulation of GCH1.
genome sequencing data (Lu et al., 2016), for a final sample
size of 83. DISCUSSION
We identified a total of 384 GCH1 sequence variants (SNPs)
in the 83 unrelated Tibetans, among which 245 were shared Hypoxia serves as a key stress in high altitude environments.
between Tibetans and the three lowland reference populations For lowlanders, prolonged exposure to high altitude hypoxia
from the 1000 Genomes Project (http://www.1000genomes.org) can cause chronic mountain sickness, reflected by the over
(103 Han Chinese, CHB; 99 Europeans, CEU and 108 Africans, production of red blood cells (polycythemia) as well as other
YRI). The remaining 139 SNPs were rare in Tibetans (<1.0%), deleterious physiological changes (Hackett & Roach, 2001;
and therefore not included in our analysis. The 245 SNPs were Macinnis et al., 2010). Tibetans are genetically adapted to high
all located in the non-coding regions of GCH1. altitude hypoxia, and exhibit blunted physiological responses,
To detect whether GCH1 was under selection in Tibetans, we e.g., relatively low hemoglobin levels (Lorenzo et al., 2014;
conducted four different tests of selection, including two allele- Peng et al., 2011, 2017; Xiang et al., 2013). Key hypoxic
frequency-based tests (F and Tajima’s D) and two haplotype- pathway genes EPAS1 and EGLN1 were reported to be
ST
based tests (iHS and XP-EHH). We identified 49 GCH1 SNPs responsible for these blunted physiological responses (Lorenzo
with large between-population (Tibetan vs. Han) divergence et al., 2014; Peng et al., 2017; Xiang et al., 2013). However,
(F >0.2), much larger than the genome average (F =0.03). while the Tibetan version of these two genes provide protection
ST ST
These high-F variants also showed high iHS and XP-EHH against polycythemia, they do not explain all physiological
ST
values (iHS>0.2 and/or XP-EHH>0.2) (Figure 1). They were changes in Tibetans, suggesting there might be other genes
aggregated in a relatively short region (7.5 kb) covering intron-1 involved given the complexity of high altitude adaptation.
and intron-2 of GCH1. These results suggest a clear signal of Based on resequencing and population analysis, we
positive Darwinian selection on GCH1 in Tibetans (Supplementary confirmed a signal of selection on GCH1 in Tibetans. We
Table S1) identified more than 40 variants showing deep allelic divergence
between highlander Tibetans and lowlander Han Chinese, with
Functional prediction and genetic association analysis some having potential functional effects based on prediction
To determine the potential function of the SNPs with selection using ENCODE. GCH1 is a rate-limiting enzyme, acting as a
signals, we performed functional predictions using evolutionary crucial factor for maintaining normal NO synthetase function
constraint (GERP), transcription factor binding sites (TFBS), and blood pressure. Inhibition of GCH1 activity is related to
splicing motif, H3K4Me1/H3K4Me3 sites, and DNase-I several cardiovascular diseases, with GCH1 found to prevent
hypersensitive sites. Results showed that there might be hypoxia-induced pulmonary hypertension (Khoo et al., 2005).
multiple functional sites, reflected by the consistent signals of Hence, the function of selection on GCH1 in Tibetans is expected
different functional predictions (Supplementary Table S1). to help maintain proper cardiovascular function at high altitude.
To test whether these candidate GCH1 variants contribute to Hypoxic pulmonary vasoconstriction and pulmonary vascular
the adaptive physiological traits of Tibetans, we measured structural remodeling are dominant pathophysiological
blood NO concentrations, SaO levels, and Hb concentrations characteristics of hypoxic pulmonary hypertension (Galiè et al.,
2
in 226 unrelated adult Tibetans (refer to methods for details). 2016; McLaughlin & McGoon, 2006; Schermuly et al., 2011).
For genotyping, we selected nine tag SNPs to represent the When lowlanders move to high altitudes, pulmonary
entire gene region of GCH1. Notably, four SNPs (rs7148266, hypertension usually occurs within a few weeks (Wilkins et al.,
rs17128004, rs4411417, and rs10136972) showed significant 2015; Wu & Kayser, 2006); however, Tibetans rarely develop
association with blood NO levels, with the adaptive alleles this condition. We showed that GCH1 SNPs were associated
exhibiting decreased NO. Each SNP accounted for 1.88%– with NO levels in the blood. GCH1 is involved in the synthesis
3.76% of NO variance (Figure 2). Similar results were observed of tetrahydrobiopterin (BH4), a vital regulator of eNOS, the
when males and females were analyzed separately (Table 1). endothelial-form enzyme producing NO, an important molecule
Additionally, two SNPs (rs146540091 and rs137863726) for vasodilation, which is considered the main reason for the
showed association with hemoglobin concentration, with the superior blood flow and pulmonary pressure in Tibetans (Pickert
158 www.zoores.ac.cn
Zoological Research 38(3): 155-162, 2017 159
Figure 2 Genetic association of seven GCH1 variants with three physiological traits (blood NO concentration, blood oxygen saturation
level, and hemoglobin concentration)
160 www.zoores.ac.cn
et al., 2013). In hph-1 mice, deficiency of BH4 causes hypoxia- Ernst J, Kellis M. 2012. ChromHMM: automating chromatin-state discovery
induced pulmonary hypertension even under normoxic and characterization. Nature Methods, 9(3): 215-216.
conditions (Khoo et al., 2005). The overexpression of GCH1 in
Erzurum SC, Ghosh S, Janocha AJ, Xu W, Bauer S, Bryan NS, Tejero J,
mice could prevent hypoxia-induced pulmonary hypertension
Hemann C, Hille R, Stuehr DJ, Feelisch M, Beall CM. 2007. Higher blood
due to the augmentation of BH4 (Khoo et al., 2005). Hence, it is
flow and circulating NO products offset high-altitude hypoxia among
possible that GCH1 regulates pulmonary vasoconstriction
Tibetans. Proceedings of the National Academy of Sciences of the United
responses in Tibetans by influencing NO production in the blood.
States of America, 104(45): 17593-17598.
We observed four GCH1 variants showing significant
Galiè N, Humbert M, Vachiery JL, Gibbs S, Lang I, Torbicki A, Simonneau G,
association with blood NO levels. As these variants are located
Peacock A, Noordegraaf AV, Beghetti M, Ghofrani A, Sanchez MAG,
in the GCH1 intron regions with peak enhancer and/or promoter
Hansmann G, Klepetko W, Lancellotti P, Matucci M, McDonagh T, Pierard
activity signals, they are likely involved in the regulation of
LA, Trindade PT, Zompatori M, Hoeper M, Aboyans V, Vaz Carneiro A,
GCH1 expression and eventually affect blood NO production,
Achenbach S, Agewall S, Allanore Y, Asteggiano R, Badano LP, Barberà JA,
which needs further investigation. We also observed
Bouvaist H, Bueno H, Byrne RA, Carerj S, Castro G, Erol C, Falk V, Funck-
associations of GCH1 SNPs with oxygen saturation and
Brentano C, Gorenflo M, Granton J, Iung B, Kiely DG, Kirchhof P, Kjellstrom
hemoglobin; however, the underlying molecular mechanisms
B, Landmesser U, Lekakis J, Lionis C, Lip GY, Orfanos SE, Park MH,
are yet to be studied.
Piepoli MF, Ponikowski P, Revel MP, Rigau D, Rosenkranz S, Völler H,
In summary, we demonstrated that GCH1 has been under
Zamorano JL. 2016. 2015 ESC/ERS Guidelines for the diagnosis and
positive selection in Tibetans. We identified many variants with
treatment of pulmonary hypertension: the joint task force for the diagnosis
deep allelic divergence between Tibetans and lowlanders. The
and treatment of pulmonary hypertension of the european society of
association and known function results suggest the potential
cardiology (ESC) and the european respiratory society (ERS): endorsed by:
involvement of GCH1 in the regulation of multiple physiological
association for european paediatric and congenital cardiology (AEPC),
traits in Tibetans.
international society for heart and lung transplantation (ISHLT). European

Heart Journal, 37(1): 67-119.
ACKNOWLEDGEMENTS
Hackett PH, Roach RC. 2001. High-altitude illness. The New England
We are grateful to all the volunteers participated in this study. Journal of Medicine, 345(2): 107-114.

Khoo JP, Zhao L, Alp NJ, Bendall JK, Nicoli T, Rockett K, Wilkins MR,
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